For applications such as protein screening, organic and inorganic molecular development, and pre-screening of toxins and other molecules for sensor applications, there is a need for nanoporous filters and screens with uniform pore size, scalable between ˜1-100 nm. For example, all protein production, isolation and purification efforts require that the proteins be: (a) separated away from other contaminating proteins and other molecules, (b) analyzed to assess its degree of homogeneity, and (c) treated to change the type of solution or buffer in which it is dissolved. The fact that each protein behaves differently in each of these steps can often make the task of working with isolated proteins difficult, particularly when the goal is to develop high throughput methods for their production and purification. Differences in homogeneity following purification can be caused by variation in post-translational modifications, dissociation of subunits, differences in the degree of folding, and proteolytic degradation.
A variety of methods have been developed for determining the size of a protein or protein complex, assessing the heterogeneity of the population, or separating proteins from other molecules. For example, conventional methods for assessing the size of protein complexes have included size exclusion chromatography (SEC) or electrophoresis in native gels, dynamic light scattering, electron microscopy, scanning probe microscopy, sedimentation rates, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, neutron scattering, and small angle X-ray scattering (SAXS). In addition to providing an estimate of size, several of these methods (SEC, sedimentation, dynamic light scattering) have been used to directly or indirectly facilitate protein separation and purification. The development of many of these standard methods for high-throughput applications in microfabricated systems, however, has remained difficult. Most methods currently used in high-throughput, chip-based systems involve electrophoretic separations of the components. Many of these techniques also require costly instrumentation and are labor intensive.
And some common methods for characterizing the homogeneity of a bio-molecule such as a protein or toxin are those that separate the components based on physical size (e.g. size exclusion chromatography, mass spectrometry) or a combination of size and charge density (e.g. gel electrophoresis). While all three techniques can be adapted for high throughput applications and incorporated into automated systems, each has limitations. For example, size exclusion chromatography using gel matrices dilutes the sample and has limited resolving power to provide accurate details about size heterogeneity. Mass spectrometry can provide the most accurate assessment of sample homogeneity, but variations in ionization efficiency can make it difficult to accurately quantify the relative proportion of the components. And electrophoretic methods can resolve molecules that differ by as little as a single positive or negative charge, but apparently homogeneous samples can often contain multiple components that have the same charge density per unit mass.
Advances in the development of silicon and other materials with nanometer-scale (1-1000 nm) pores or slits have raised the possibility of producing molecule sizing filters with a sufficiently large dynamic range of size selection (extending from ˜1 nm to 1 μm) to cover, for example, the entire range of known sizes of proteins and protein complexes. However, the use of standard lithographic processes for producing the smallest of these features sizes (i.e. in the range of ˜1-100 nm) has been difficult for large areas (i.e. greater than 1 cm2, and typically in the range of tens of cm2) required for most molecular filter applications. And while non-lithographic methods have been developed for producing near-nanometer pore sizes, their usefulness is limited due to lack of pore size uniformity and repeatability. For example, porous membranes created through anodic etching and mesoporous silica formed through sol-gel process have non-uniform pore diameters, respectively, which typically vary over a broad range: ˜30-400 nm for anodic alumina and ˜2-20 nm in sol-gel films. These limitations are difficult to address due to critical dependence of process chemistry on several variables such as solution concentration, temperature, and current. Other filter materials such as zeolites have uniform pores, but only in the relatively narrow range of ˜0.3-3 nm. Carbon nanotubes are being developed at the Lawrence Livermore National Laboratory for filters in the 1-10 nm range, but scaling beyond this limit is extremely challenging, and the cylindrical shape of the pores may present additional complications. Finally, ion-track etching through polycarbonate films can produce a wide range (−10 nm to ˜μm) of pore diameters, but pore uniformity and flow rates have been observed to be limited to about ±20% and <0.1 mL/min/cm2, respectively, for 10 nm diameter pores.
Thus there is a need for a method of fabricating large-area nanoporous filters and screens having uniform pores with scalable pore diameters ranging from a few nanometers to hundreds of nanometers, and capable of efficiently separating and characterizing molecules and small particles.